Am J Physiol Regul Integr Comp Physiol 304: R333–R342, 2013.
First published December 19, 2012; doi:10.1152/ajpregu.00409.2012.
High-intensity interval training increases in vivo oxidative capacity with no
effect on Pi¡ATP rate in resting human muscle
Ryan G. Larsen,1 Douglas E. Befroy,2 and Jane A. Kent-Braun1
1
Department of Kinesiology, University of Massachusetts, Amherst, Massachusetts; and 2Department of Diagnostic Radiology,
Yale University School of Medicine, New Haven, Connecticut
Submitted 6 September 2012; accepted in final form 19 December 2012
mitochondria; oxidative metabolism; phosphorus magnetic resonance
spectroscopy; cycling exercise
to endurance exercise training is an
increased capacity to supply ATP via oxidative phosphorylation in skeletal muscle. This adaptation occurs in response to
increased mitochondrial content and is associated with improved exercise capacity (31, 32). In contrast, little is known
about the effect of exercise training on oxidative ATP production in resting muscle, which accounts for a significant portion
of daily energy expenditure (55), and therefore may be important for overall metabolic health. Thus, while there is some
evidence to suggest an association between mitochondrial
content and ATP turnover in resting muscle (33, 57), it is not
clear whether the exercise-induced adaptations responsible for
increased oxidative capacity may also result in increased ATP
production at rest. Furthermore, there is limited information on
the scope and timing of exercise-induced adaptations in mitochondrial ATP production in vivo, which could have important
A FUNDAMENTAL ADAPTATION
Address for reprint requests and other correspondence: J. A. Kent-Braun,
Dept. of Kinesiology, Univ. of Massachusetts, 108 Totman Bldg., 30 Eastman
Lane, Amherst, MA 01003 (e-mail: [email protected]).
http://www.ajpregu.org
implications for developing training interventions designed to
enhance oxidative metabolism in resting and exercising skeletal muscle.
The effects of high-volume, endurance exercise training on
oxidative metabolism in skeletal muscle were first demonstrated more than 40 years ago (31). More recently, studies
from several laboratories have shown that low-volume, highintensity, interval-style training (HIT) also is an effective
approach for promoting mitochondrial biogenesis (9 –11, 19,
21, 45, 62). In fact, studies have reported increased expression
of peroxisome proliferator-activated receptor ␥ coactivator 1␣
(PGC-1␣), which is an important factor for coordinating the
activation of genes encoding mitochondrial proteins, after a
single session of exercise training (45, 46, 68). Similarly, some
reports have shown that markers of in vitro oxidative capacity
can be increased after a single session of high-intensity exercise (18, 63). Together, this group of studies has reformed the
conventional notion that long-term, high-volume training interventions are required to stimulate mitochondrial biogenesis.
However, despite numerous investigations of the molecular
events accompanying HIT, the time course for functional
changes in muscle oxidative capacity in vivo is still unclear.
Mitochondrial ATP production is vital for meeting cellular
energy demand not only during periods of high ATP turnover
but also in resting muscle (56). Because resting energy expenditure accounts for ⬃60 –75% of total daily energy expenditure, ATP flux in resting skeletal muscle may play an important
role in body weight maintenance, as well as regulation of
glucose and lipid metabolism (43, 50, 51, 69). Whereas numerous energy-consuming processes are active in resting tissue, protein synthesis is believed to account for a large portion
of ATP turnover in resting skeletal muscle (55, 56). Various
modes of exercise training have been shown to elevate rates of
muscle protein synthesis and breakdown (4, 5, 52, 67), suggesting that exercise may result in elevated ATP demand in
resting tissue, which presumably would be reflected by increased oxidative ATP flux.
The rate of ATP synthesis in resting tissue has been estimated using the saturation transfer experiment, which quantifies the rate of Pi incorporation into ␥-ATP, i.e., VPi¡ATP (2,
37, 50, 64). Notably, in part due to glycolytic contributions,
VPi¡ATP overestimates oxidative ATP synthesis in resting
muscle compared with other measures of aerobic energy metabolism (20, 38), which may limit the ability of the saturation
transfer experiment to detect changes in oxidative ATP production in resting muscle. Despite this potential limitation,
several studies have shown elevated VPi¡ATP in response to
various physiological stimuli, e.g., hyperglycemia (44) and
hyperinsulinemia (6, 51), suggesting that the saturation transfer
technique is sensitive to changes in overall ATP turnover.
0363-6119/13 Copyright © 2013 the American Physiological Society
R333
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Larsen RG, Befroy DE, Kent-Braun JA. High-intensity interval
training increases in vivo oxidative capacity with no effect on
Pi¡ATP rate in resting human muscle. Am J Physiol Regul Integr
Comp Physiol 304: R333–R342, 2013. First published December
19, 2012; doi:10.1152/ajpregu.00409.2012.—Mitochondrial ATP
production is vital for meeting cellular energy demand at rest and
during periods of high ATP turnover. We hypothesized that highintensity interval training (HIT) would increase ATP flux in resting
muscle (VPi¡ATP) in response to a single bout of exercise, whereas
changes in the capacity for oxidative ATP production (Vmax) would
require repeated bouts. Eight untrained men (27 ⫾ 4 yr; peak oxygen
uptake ⫽ 36 ⫾ 4 ml·kg⫺1·min⫺1) performed six sessions of HIT (4–6 ⫻
30-s bouts of all-out cycling with 4-min recovery). After standardized
meals and a 10-h fast, VPi¡ATP and Vmax of the vastus lateralis muscle
were measured using phosphorus magnetic resonance spectroscopy at
4 Tesla. Measurements were obtained at baseline, 15 h after the first
training session, and 15 h after completion of the sixth session.
VPi¡ATP was determined from the unidirectional flux between Pi and
ATP, using the saturation transfer technique. The rate of phosphocreatine recovery (kPCr) following a maximal contraction was used to
calculate Vmax. While kPCr and Vmax were unchanged after a single
session of HIT, completion of six training sessions resulted in a ⬃14%
increase in muscle oxidative capacity (P ⱕ 0.004). In contrast, neither
a single nor six training sessions altered VPi¡ATP (P ⫽ 0.74). This
novel analysis of resting and maximal high-energy phosphate kinetics
in vivo in response to HIT provides evidence that distinct aspects of
human skeletal muscle metabolism respond differently to this type of
training.
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EFFECTS OF INTERVAL TRAINING ON MUSCLE ENERGETICS IN HUMANS
METHODS
Participants. Eight healthy young males (27.0 ⫾ 3.4 yr, 176.1 ⫾
6.9 cm; 83.0 ⫾ 15.4 kg; means ⫾ SE) volunteered for the study. A
preliminary screening session was used to ensure that all participants:
1) had no history of neurological, pulmonary, cardiovascular, or
metabolic disease; 2) were nonsmokers and able to undergo magnetic
resonance procedures (no metal implants); 3) did not participate in
any type of regular exercise; 4) did not take any medication or dietary
supplements shown to affect muscle function or metabolism; and
5) had no first-degree relatives with Type 2 diabetes to eliminate
potential differences in the metabolic response to exercise training.
All experimental procedures and potential risks associated with the
study were explained to the volunteers, who provided written informed consent, as approved by the institutional review boards at the
University of Massachusetts, Amherst and Yale University School of
Medicine.
Preparatory Procedures
Participants came into the laboratory for a preparatory session 7 to
14 days before the start of the training intervention. Measures of
height, body mass, and blood pressure were obtained. Participants
practiced maximal voluntary isometric contractions (MVICs) of the
knee extensor muscles to familiarize them with the contraction protocol that would be used for muscle metabolic testing at Yale University. As described previously (41), participants were positioned
supine on a patient bed, with the knee fixed at 35° from straight over
a custom-built apparatus with a built-in strain gauge. The foot was
held down with a cushioned strap placed over the ankle joint, which
allowed for an isometric contraction of the knee extensor muscles and
minimal movement of the limb. This setup matched the arrangement
that was used in the 4-Tesla MR system at Yale University. Participants practiced MVICs (3–5 s duration, 2 min recovery between each)
to ensure that these contractions could be performed consistently.
Next, participants performed an incremental exercise test to voluntary exhaustion on a mechanically braked cycle ergometer (828E,
Monark, Vansbro, Sweden) to determine peak, whole body, oxygen
consumption (V̇O2 peak). Respiratory gases from the expired air were
measured using a metabolic cart (TrueOne 2400, ParvoMedics,
Sandy, UT), and the highest value for oxygen consumption achieved
over a 15-s collection period was used as V̇O2 peak. Subsequently,
participants performed a 30-s sprint on the cycle ergometer (i.e.,
Wingate Test) to become familiarized with the training protocol.
Three days after completing the final session of the training protocol,
the participant returned to the laboratory to repeat the incremental
V̇O2 peak test.
To quantify habitual physical activity level at baseline, participants
were instructed in the use of a uniaxial accelerometer (GT1M,
Actigraph, Pensacola, FL) (41). The accelerometer was worn at the
waist for 7 consecutive days during all waking hours, while usual
physical activity routines were maintained. Average daily counts were
recorded to characterize the study group and confirm their relatively
sedentary usual behavior.
Muscle Metabolic Testing
Participants were transported to the Magnetic Resonance Research
Center at Yale University to determine the effects of HIT on skeletal
muscle metabolism using 31P-MRS. Before and during these visits,
participants were provided standardized meals, as described below.
The muscle metabolic testing sessions were conducted three times:
1) 9 h before the first training session (baseline), 2) 15 h after the first
training session (15 h post), and 3) 15 h after completing the sixth
training session (2 wk post). Each testing session included measurements of 1) intracellular pH and concentrations of relevant energy
metabolites, 2) unidirectional flux from Pi to ATP, 3) apparent
longitudinal relaxation time of Pi (T1=), and 4) PCr recovery following
a 24 s MVIC. Measurements of pH and metabolite concentrations in
resting muscle and Pi¡ATP flux were acquired after a 10-h overnight
fast and lasted approximately 2 h. Participants were then given a light
snack (10% of estimated daily energy expenditure) and allowed a
short break outside the scanner before performing the muscle contraction and PCr recovery protocol.
All 31P-MRS measurements were performed on a 4-Tesla whole
body system (Bruker Biospin, Ettlingen, Germany) and acquired with
an MR probe (31P surface coil: 6 ⫻ 8 cm, 1H surface coil: 9 cm
diameter) placed over the VL muscle of the dominant leg. To ensure
consistent positioning of the MR probe across all testing sessions, the
probe location was marked with a pen on the participant’s thigh. The
participant was positioned on the patient bed and centered in the magnet.
A series of axial plane scout images were first acquired to ensure
optimal positioning of the VL in the isocenter of the magnet. In
addition, the image slice corresponding to the center of the MR probe
was used for measuring VL cross-sectional area (cm2) using Image J
software (http://rsb.nih.gov/ij/). Magnetic field homogeneity was optimized by localized shimming on the proton signal from tissue water
using the 1H coil (41).
Intracellular pH and metabolites in resting muscle. To ensure
accurate estimation of intracellular 31P metabolites and pH in resting
muscle, each testing session started with collection of a fully relaxed
spectrum (repetition time ⫽ 35 s, 16 averages) acquired with an
adiabatic excitation pulse and 90° flip angle. From the fully relaxed
spectrum, concentrations of intracellular metabolites were determined
based on the assumptions that total creatine ([PCr] ⫹ [creatine]) ⫽
42.5 mM, and free creatine ⬵ Pi in skeletal muscle (28, 48). To verify
potential changes in metabolite concentrations, we also determined
31
P metabolite ratios that provided insight about exercise-induced
changes in the intracellular milieu with potential influence on enzy-
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To date, only a few studies have investigated the effects of
exercise training on ATP production in resting human muscle
(37, 64). Kacerowsky-Bielesz et al. (37) showed that VPi¡ATP
in the gastrocnemius-soleus complex increased after only three
sessions of moderate-intensity cycling, whereas Trenell et al.
(64) showed no effects on VPi¡ATP after 2 or 8 wk of daily
walking. In a cross-sectional study, Befroy et al. (3) reported
similar VPi¡ATP in the calf muscles of untrained and endurance-trained young men, suggesting that oxidative ATP flux in
resting muscle is not different in chronically trained muscle.
One explanation for these contrasting results is that resting
ATP turnover may increase transiently at the onset of training
and then return to a steady-state rate in chronically trained
muscle. Currently there are conflicting results in the literature
regarding a potential relationship between resting and maximal
rates of ATP synthesis in skeletal muscle (57, 64, 65). To date,
no studies have examined the timing and magnitude of exercise-induced changes in both resting and maximal rates of
oxidative ATP production.
The purpose of this study was to examine the acute and
short-term effects of HIT on human skeletal muscle energetics
in vivo using phosphorus magnetic resonance spectroscopy
(31P-MRS). The vastus lateralis (VL) muscle of young, untrained men was studied before, during, and after a training
period of high-intensity cycling exercise. We hypothesized that
1) a single session of HIT would increase VPi¡ATP, with no
change in oxidative capacity (Vmax), and 2) six bouts of HIT
would increase Vmax with no further change in VPi¡ATP. The
results provide new and unique information regarding the time
course and magnitude of changes in distinct aspects of oxidative metabolism in human skeletal muscle after HIT.
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EFFECTS OF INTERVAL TRAINING ON MUSCLE ENERGETICS IN HUMANS
VPi¡ATP ⫽ k⬘ · 关Pi兴
Control
saturation
10
γ-ATP
saturation
Pi
5
0
-5
-10
-15 PPM
matic reactions in resting skeletal muscle. Intracellular pH was calculated based on the chemical shift between the Pi and PCr resonances
(49). Because the concentration of ADP is too low to be detected in
the spectrum, free intracellular [ADP] was calculated, according to the
creatine kinase equilibrium:
关ADP兴free ⫽ 关ATP兴关Pi兴 ⁄ 共kCK 关H⫹兴关PCr兴兲
with the assumption of equal concentrations of free creatine and Pi in
skeletal muscle (48). The equilibrium constant of the creatine kinase
reaction (kCK) was assumed to be 1.66·109 M⫺1 (66).
Resting oxidative ATP production. The Pi¡ATP rate in resting
skeletal muscle (VPi¡ATP) was determined using the 31P saturation
transfer experiment (2, 7). Steady-state magnetization of Pi (M=) was
measured in the presence of selective saturation of the ␥-ATP resonance. A control spectrum was also acquired to yield equilibrium
magnetization of Pi (M0) in the absence of ␥-ATP saturation. To
compensate for off-resonance effects of the saturation pulse, the
control spectrum was acquired with the saturation pulse centered on a
downfield frequency equidistant from Pi (Fig. 1). Additional parameters were 1-ms adiabatic half-passage excitation pulse (centered
between Pi and ␥-ATP), 15-s continuous low-power saturation pulse,
sweep width of 10,000 Hz, 2,048 complex data points, 15-s repetition
time, and 16 averages. Two sets of these paired 31P spectra were
acquired and line-fit, and the results were then averaged.
The ratio of steady state relative to equilibrium magnetization of Pi
under conditions of ␥-ATP saturation (M=/M0) is given by the equation:
Spectral Analysis
All MRS data were processed using NUTS software (Acorn NMR,
Livermore, CA). Free induction decays were zero filled to 16 k points
and multiplied by an exponential factor corresponding to 10 Hz
line-broadening before Fourier transformation. The 31P spectra were
phased and baseline was corrected before peaks corresponding to PCr,
Pi, and ␥-ATP were fit to Lorentzian line shapes. All MRS analyses
were performed by the same investigator who was blinded to the
identity of the data. The data were averaged to yield 31P spectra with
temporal resolution of 1 min at rest, 4 s during the MVIC, 8 s during
first 5 min of recovery, and 30 s during last 5 min of recovery (Fig. 2).
The recovery data, from the end of the 24-s MVIC through 10 min
of recovery, were fitted with a three-parameter monoexponential
equation to determine the rate constant kPCr:
PCr 共t兲 ⫽ ⌬PCr共1 ⫺ e⫺kPCr·t兲 ⫹ PCrend
where PCrend is the integral of PCr at the end of the MVIC, and ⌬PCr
is the difference in PCr between rest and end of the MVIC (41, 48).
According to the linear model of muscle respiration (48), the maximal
rate of oxidative ATP production can be estimated as the product of
kPCr and resting concentration of PCr, using the following equation:
Vmax共mM ATP · s⫺1兲 ⫽ kPCr · 关PCr兴rest
PCr
ATP
γ
M⬘⁄ M 0 ⫽ 1 ⁄ 共1 ⫹ k⬘· T1兲
where k= is a pseudo first-order rate constant describing the loss of Pi
magnetization due to exchange of saturated spins between Pi and
ATP, and T1= is the apparent longitudinal relaxation time of Pi in the
presence of continuous ␥-ATP saturation. Calculation of T1= was
accomplished using a seven-point inversion recovery experiment
(Sigmaplot, Systat Software) with multiple delays (0.05, 1.2, 3.1, 6,
10, 15, 20 s; 16 averages) between the adiabatic 180° inversion pulse
and the 90° detection pulse (2). After determining T1= and M=/M0, the
exchange rate constant of Pi¡ATP was calculated based on the
equation:
k⬘ ⫽ 关1 ⫺ M⬘/M 0兴 ⁄ T1
and the unidirectional rate of oxidative ATP synthesis was calculated
as the product of k= and [Pi] (2, 7):
β
α
Pi
10
10 min
recovery
5
0
-5
-10
-15
PPM
24 s
MVIC
Fig. 2. Stackplot of phosphorus spectra acquired from the vastus lateralis
muscle of a representative subject during the muscle contraction protocol. The
24-s maximal voluntary isometric contraction depleted phosphocreatine (PCr)
to ⬃60% of resting concentration. The rate constant for PCr recovery (kPCr)
was determined based on a monoexponential fit of the data during 10 min of
recovery.
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Fig. 1. Phosphorus spectra acquired from vastus lateralis muscle during the
saturation-transfer experiment. Selective ␥-ATP saturation (arrow, right) is
shown in green, with the control spectrum in blue. The control spectrum had
selective saturation (arrow, left) at a downfield frequency equivalent to the
frequency separation between ␥-ATP and Pi. All other data acquisition parameters were the same in both conditions. The ratio of Pi magnetization with and
without saturation (M=/M0) reflects the exchange of saturated spins from Pi to
ATP and is used to calculate the unidirectional Pi¡ATP flux.
Maximal oxidative ATP production. For the muscle contraction
protocol, participants were positioned on the patient bed with the knee
fixed at 35° of flexion over a custom-built apparatus, as described for
the preparatory procedures and done previously (41). To standardize
conditions, participants performed two brief “warm-up” MVICs
(3–5 s duration, 2 min rest between each) before performing two 24-s
MVICs contractions, separated by 10 min of recovery. The contraction duration of 24 s was selected as it has been shown to deplete PCr
to ⬃50 –70% of resting levels in VL, without inducing significant
acidosis (41, 42). The average of the two trials was calculated for each
variable of interest. Participants were provided verbal encouragement
and visual force feedback (from a series of light-emitting diodes)
during all contractions. Free induction decays with 2,048 complex
data points were acquired continuously for 11 min, with a nominal 60°
hard pulse, 2 s repetition time, and spectral width of 8,000 Hz.
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EFFECTS OF INTERVAL TRAINING ON MUSCLE ENERGETICS IN HUMANS
Table 1. Group characteristics at baseline
Age, yr
Height, m
Body mass, kg
Systolic blood pressure, mmHg
Diastolic blood pressure, mmHg
Physical activity, daily counts/1,000
27.0 ⫾ 3.6
1.76 ⫾ 0.07
83.0 ⫾ 15.4
126.8 ⫾ 4.9
77.5 ⫾ 3.2
294.3 ⫾ 37.1
Data are from 8 males and are expressed as means ⫾ SE.
Dietary and Physical Activity Controls
RESULTS
Participant Characteristics
Descriptive characteristics of the participants are presented
in Table 1. The accelerometer data provide a measure of
habitual physical activity, and the average daily counts reported are consistent with other studies of relatively sedentary
young adults (40, 41). In agreement with these data, average
V̇O2 peak was 35.8 ⫾ 4.0 ml·min⫺1·kg⫺1 at baseline and improved to 39.3 ⫾ 4.5 ml·min⫺1·kg⫺1 (P ⫽ 0.03) after completion of the 2-wk training period.
The total workload performed during the six training sessions was 431.4 ⫾ 26.9 kJ, which is consistent with training
data (⬃225 kJ/wk) in a previous HIT study (10). In addition,
mean (156.3 ⫾ 1.0 beats/min, ⬃86.2% of maximal) and peak
(180.5 ⫾ 1.3 beats/min, ⬃99.5% of maximal) heart rate
achieved during all thirty 30-s training bouts suggested maximal effort of the participants. Baseline cross-sectional area of
the VL muscle (23.24 ⫾ 2.47 cm2) was comparable to that
reported by Hulmi et al. (34) in young men and did not change
after the first (23.06 ⫾ 2.13 cm2) nor the sixth training sessions
(24.36 ⫾ 2.21 cm2; P ⫽ 0.79).
Metabolite Levels in Resting Muscle
Training Protocol
The training protocol consisted of six sessions of cycling over a
2-wk period, with each training session performed on the same
ergometer that was used for the V̇O2 peak test. Participants performed
three training sessions per week, with a minimum of 36 h between
sessions (week 1: Saturday, Tuesday, Friday; week 2: Monday,
Wednesday, Friday). Each training session started with a 5 min
warm-up at 60 W, including two brief sprints (4 –5 s duration). This
was followed by 4 – 6 bouts of 30 s “all-out” effort. The wheel
resistance [7.5% of body mass, (11)] was adjusted manually by one of
the investigators, and the participant was instructed to reach maximal
effort within the time frame (⬃2–3 s) required to arrive at the
appropriate resistance. Each 30-s bout was followed by 4 min of
active recovery, during which the participant pedaled against a light
resistance to minimize venous pooling and any feeling of lightheadedness (10). Consistent with previous studies using this HIT
protocol, sessions 1 and 2 consisted of four bouts, the next two
sessions comprised five bouts, and participants performed six bouts
for sessions 5 and 6.
Because a primary objective of the study was to examine the effects
of a single session of HIT on skeletal muscle energetics, the first
training session was performed at the Magnetic Resonance Research
Center at Yale University, where the metabolic testing was conducted.
The subsequent five sessions were performed at the University of
Massachusetts, Amherst. All training sessions were supervised by the
same two investigators, who provided verbal encouragement, and
continuously monitored heart rate and general well being of the
participants.
Statistical Analyses
Comparison of V̇O2 peak, before and after training, was accomplished by Student’s paired t-test. The effects of training on muscle
metabolite levels and rates of skeletal muscle ATP synthesis were
evaluated across the three testing sessions using mixed-model, repeated measures ANOVAs with unstructured covariance structure.
The effects of training on metabolite levels in resting
muscle are summarized in Table 2. Intracellular [Pi] increased after the first training session (P ⫽ 0.005) and
remained elevated at the end of the 2-wk protocol (P ⬍ 0.001),
with no difference between the second and sixth training
sessions (P ⫽ 0.28). Concomitantly, [PCr] decreased from
baseline after the first and remained lower after the final
training sessions. Intracellular pH in resting muscle increased
after the first training session (P ⫽ 0.04) and was further
elevated after 2 wk of training (P ⫽ 0.002). Cytosolic [ATP]
was reduced after the first training session (P ⬍ 0.001), with a
further decline after the sixth session (P ⬍ 0.001, Fig. 5).
Relative to baseline, [ADP]free was lower after 2 wk of training
(P ⫽ 0.02).
These changes in metabolite concentrations were reflected in
the ratios of specific metabolite pairs. Specifically, the Pi/PCr
ratio increased after the first training session (P ⫽ 0.005) and
Table 2. Metabolite levels and pH in resting muscle
PCr, mM*†
Pi, mM*†
ATP, mM*†‡
ADP, ␮M†
Pi/PCr*†
PCr/ATP*†‡
ADP/ATP, 103*†
pHrest*†‡
Baseline (1)
15 h post (2)
2 wk post (3)
P Value
37.38 ⫾ 0.22
5.12 ⫾ 0.22
8.87 ⫾ 0.33
8.72 ⫾ 0.69
0.14 ⫾ 0.01
4.26 ⫾ 0.17
0.97 ⫾ 0.05
7.07 ⫾ 0.00
36.77 ⫾ 0.21
5.73 ⫾ 0.21
6.98 ⫾ 0.29
7.88 ⫾ 0.67
0.16 ⫾ 0.01
5.34 ⫾ 0.23
1.12 ⫾ 0.05
7.08 ⫾ 0.01
36.58 ⫾ 0.21
5.92 ⫾ 0.21
6.06 ⫾ 0.26
7.26 ⫾ 0.60
0.16 ⫾ 0.01
6.11 ⫾ 0.26
1.21 ⫾ 0.05
7.09 ⫾ 0.01
0.001
0.001
⬍0.001
0.07
⬍0.001
⬍0.001
⬍0.001
0.004
Data are from the vastus lateralis muscle of 8 males and are expressed as
means ⫾ SE. P values for main effects from repeated measures ANOVAs are
shown, and statistical differences (P ⬍ 0.05) across time points from post hoc
analyses are indicated by * (1 vs. 2), † (1 vs. 3), ‡ (2 vs. 3).
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Participants were instructed to maintain their usual diet and continue their regular physical activity routines during the study. In
addition, dietary intake and physical activities were standardized
before each muscle metabolic testing session to minimize potential
confounding effects on our measures of muscle energetics. Specifically, the muscle metabolic testing sessions were performed at the
same time of day across trials, and participants were asked to avoid
strenuous activities (expect for the training session) for 48 h before
each test session. Dietary intake before each study was controlled by
providing participants with a standardized meal (30% of estimated
daily energy expenditure) and a snack (10% of estimated daily energy
expenditure) 11 and 10 h before the testing sessions, respectively. The
snack was provided to minimize hunger during the MRS measures the
next morning. Macronutrient composition (⬃60% carbohydrate,
⬃25% fat, ⬃15% protein) was similar for all participants and consistent across trials. Daily energy expenditure was estimated based on
the Harris-Benedict Equation (27) and multiplied by an activity factor
of 1.5.
Post hoc analyses were done using pairwise comparisons (Student’s
paired t-test), and exact P values were reported. Differences were
considered significant when P ⬍ 0.05. All data are presented as
means ⫾ SE.
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EFFECTS OF INTERVAL TRAINING ON MUSCLE ENERGETICS IN HUMANS
Table 4. Variables from the PCr recovery experiment
Table 3. Variables from saturation transfer experiment
M=/M0, %
T1=, s*†
k=, s⫺1
Pi, mM*†
Vrest, mM/min
Baseline (1)
15 h post (2)
2 wk post (3)
P Value
0.84 ⫾ 0.01
4.23 ⫾ 0.09
0.039 ⫾ 0.003
5.12 ⫾ 0.22
11.87 ⫾ 1.14
0.85 ⫾ 0.01
4.63 ⫾ 0.09
0.032 ⫾ 0.002
5.73 ⫾ 0.21
11.01 ⫾ 0.89
0.85 ⫾ 0.01
4.57 ⫾ 0.15
0.033 ⫾ 0.001
5.92 ⫾ 0.21
11.78 ⫾ 0.43
0.53
0.003
0.22
0.001
0.74
Data are from the vastus lateralis muscle of 8 males (n ⫽ 7 for 15 h post,
because of technical issues) and are expressed as means ⫾ SE. M=/M0, ratio of
Pi magnetization with and without selective saturation of ␥-ATP; T1=, apparent
longitudinal relaxation time of Pi with saturation of ␥-ATP; k=, Pi¡ATP
exchange pseudo first-order rate constant; Vrest, resting Pi¡ATP flux. P values
are for main effect from repeated measures ANOVAs; post hoc statistical
differences (P ⬍ 0.05) between 1 and 2 (*), and 1 and 3 (†) are indicated.
Pi¡ATP Rate in Resting Muscle
2 wk post (3)
P Value
59.75 ⫾ 3.95
55.73 ⫾ 3.15
0.47
PCrend, % of rest 59.95 ⫾ 3.02
pHend*†
7.07 ⫾ 0.01
7.09 ⫾ 0.02
7.12 ⫾ 0.02 ⬍0.001
pHmin*
6.94 ⫾ 0.02
6.96 ⫾ 0.03
6.98 ⫾ 0.02
0.005
kPCr, s⫺1*†
0.0288 ⫾ 0.001 0.0278 ⫾ 0.002 0.0326 ⫾ 0.001 0.009
Vmax, mM/min*† 64.70 ⫾ 2.97
61.31 ⫾ 3.58
71.46 ⫾ 3.12
0.02
Data are from the vastus lateralis muscle of 8 males and are expressed as
means ⫾ SE. PCrend, concentration of phosphocreatine at the end of the 24-s
maximal voluntary isometric contraction; pHrest, intracellular pH at rest; pHend,
pH at the end of the 24-s MVIC; pHmin, minimum pH attained during recovery;
kPCr, rate of PCr recovery; Vmax, maximal capacity for oxidative phosphorylation. P values for main effects from repeated measures ANOVAs are shown
and statistical differences (P ⬍ 0.05) between: 1 and 3 (*) and 2 and 3 (†) are
indicated.
(coefficient of variation), M=/M0 ⫽ 2%, T1= ⫽ 6%, and
VPi¡ATP ⫽ 7%.
Muscle Contraction Protocol and Oxidative Capacity
The muscle metabolic changes observed during the contraction protocol are presented in Table 4. As designed, the 24-s
MVIC depleted PCr to ⬃50 –70% of resting concentration, and
the level of depletion was not different across the three testing
sessions (P ⫽ 0.47). Intracellular pH at the end of the 24-s
MVIC was higher after the sixth training session compared
with baseline (P ⬍ 0.001) and the first session (P ⫽ 0.009).
During the initial part of recovery, pH continued to decline and
the minimum pH reached during recovery was higher after
2 wk of training compared with baseline (P ⬍ 0.001).
Figure 4 shows individual rate constants for PCr recovery
(kPCr) acquired at baseline and after the first and sixth training
sessions. The first training session had no effect on the rate of
PCr recovery (P ⫽ 0.17), but kPCr increased by 14% after 2 wk
of training (P ⫽ 0.001, Fig. 4 and Table 4). There was good
agreement between the two measurements of kPCr performed
for each individual at each time point (coefficient of variation ⫽ 9.0%). Consistent with the kPCr data, Vmax was increased after completion of the final training session (P ⫽
0.004, Table 4). There were no correlations between kPCr or
Vmax and V̇O2 peak at baseline (r2 ⱕ 0.01, P ⱖ 0.97), or between
0.04
16
kPCr (s-1)
-1
Vrest (mM ATP·min )
The individual components contributing to the estimation of
the unidirectional rate of ATP synthesis (i.e., VPi¡ATP) in
resting muscle are summarized in Table 3. Training had no
effect on the pseudo-first-order rate constant (k=) for exchange
from Pi¡ATP (P ⫽ 0.22), VPi¡ATP (P ⫽ 0.74, Fig. 3), or
M=/M0 (P ⫽ 0.53). The apparent longitudinal relaxation time of
Pi during ␥-ATP saturation (T1=) increased after the first
training session (P ⫽ 0.001) and remained elevated after 2 wk
of training (P ⫽ 0.03). There was good agreement between the
two measurements of k= performed for each individual at each
time point (coefficient of variation ⫽ 7.9%). Data in Table 3
and Fig. 3 are the average of these two measurements.
Similarly, unpublished data from our laboratory on day-today variability of all components contributing to estimation
of VPi¡ATP show very good reproducibility, i.e., [Pi] ⫽ 4%
15 h post (2)
12
0.03
8
0.02
Baseline
15 hr post
2 week post
Fig. 3. Individual values (average of 2 measurements) from the vastus lateralis
muscle for unidirectional Pi¡ATP flux (VPi¡ATP) at baseline, 15 h after the
first training session, and 15 h after the sixth training session. The 15-h data
point for one participant (dashed line) is missing due to technical issues.
Baseline VPi¡ATP was not different from VPi¡ATP at 15 h, or after 2 wk
training (see Table 3), indicating that short-term, high-intensity interval training did not increase Pi¡ATP flux in resting muscle.
Baseline
15 hr post
2 week post
Fig. 4. Individual values (average of 2 measurements) from the vastus lateralis
muscle for the rate constant of phosphocreatine recovery (kPCr) at baseline, 15
h after the first training session, and 15 h after the sixth training session.
Baseline kPCr was unchanged after the first training session but increased after
the sixth training session (see Table 4), indicating that muscle oxidative
capacity increased with short-term, high-intensity interval training.
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remained elevated after 2 wk of training (P ⬍ 0.001), with no
difference between the second and sixth training session (P ⫽
0.28). The ratio of PCr/ATP increased after the first training
session (P ⫽ 0.002), with a further increase after the sixth
session (P ⬍ 0.001). Finally, the ADP/ATP ratio increased
after the first training session (P ⫽ 0.006) and remained
elevated at the end of the 2-wk protocol (P ⬍ 0.001), with no
difference between the second and sixth sessions (P ⫽ 0.09).
Baseline (1)
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EFFECTS OF INTERVAL TRAINING ON MUSCLE ENERGETICS IN HUMANS
11
remodeling accompanying a single session of HIT is not
adequate to enhance the capacity for oxidative ATP production
in vivo. Post hoc power calculations revealed that we were
sufficiently powered to detect an ⬃8% increase in kPCr. Thus
we believe that the design and methodology used in the present
study allowed us to detect physiologically meaningful changes
in the rate of PCr recovery.
10
ATP (mM)
9
8
7
Effects of HIT on Muscle Oxidative Capacity
6
5
0
Baseline
15 hr post
2 week post
changes in kPCr or Vmax and changes in V̇O2 peak (r2 ⱕ 0.11,
P ⱖ 0.49). While these observations suggest that V̇O2 peak was
limited by central cardiovascular factors rather than muscle
oxidative capacity, we cannot disregard the possibility that
these results were influenced by the small ranges for these
variables in the study group.
DISCUSSION
We report here for the first time the scope and timing of the
effects of HIT on bioenergetics of human skeletal muscle in
vivo. As hypothesized, the rate of PCr recovery was unchanged
after a single bout of interval training, whereas completion of
six training sessions resulted in a 14% increase in muscle
oxidative capacity. In contrast, neither a single nor six training
sessions altered the unidirectional rate of ATP synthesis, rejecting our hypothesis about increased Pi¡ATP rate in resting
skeletal muscle following HIT. These results indicate that
distinct aspects of muscle oxidative metabolism in humans
respond differently to this type of training, such that in vivo
oxidative capacity increases in response to short-term HIT,
while resting VPi¡ATP is unchanged.
Acute Effects of Exercise on Muscle Oxidative Capacity
The recent advances in our understanding of molecular
mechanisms involved in exercise-induced cellular adaptations
have provided some evidence indicating the potential for rapid
activation of mitochondrial biogenesis after exercise training
(1, 16, 32, 46, 68). While a recent study in humans reported
increased expression of PGC-1␣, mitochondrial protein content, and enzyme activities after four 30-s bouts of all-out
cycling (45), the time course for the transfer of these molecular
events into actual changes in the capacity for mitochondrial
ATP production in vivo has not been determined. Tonkonogi
and colleagues (63) showed that maximal ADP-stimulated
respiration in skinned fibers from the VL was increased by
⬃23% after a single session of high-intensity cycling, suggesting increased capacity for oxidative phosphorylation. To our
knowledge, the present study provides the first investigation of
the acute effects of a single bout of HIT on oxidative capacity
in vivo. We showed no change in the rate of PCr recovery after
the first training session (Fig. 4), indicating that the extent of
Effect of a Single Bout of HIT on VPi¡ATP
It is well established that protein turnover in skeletal muscle
can remain elevated for up to 48 and even 96 h following a
single bout of training (52, 53). Because these anabolic and
catabolic processes are coupled to ATP hydrolysis, we hypothesized that VPi¡ATP would be elevated 15 h after the first
training session due to increased overall muscle ATP turnover.
In contrast to our hypothesis, VPi¡ATP remained unchanged,
suggesting that ATP demand was not elevated after this type of
exercise training, which is consistent with the fact that [ADP],
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Fig. 5. Individual values of [ATP] from the vastus lateralis muscle at baseline,
15 h after the first training session, and 15 h after the sixth training session.
Cytosolic [ATP] was reduced from baseline after the first training session and
further reduced after the sixth training session (see Table 2).
Exercise training protocols that consist of low-volume, highintensity intervals have demonstrated potent effects on metabolic adaptations in skeletal muscle that resembles the adaptations associated with conventional, high-volume endurance
training protocols (9 –11, 21). In particular, protocols consisting of six sessions of 4 – 6 bouts of 30 s of maximal effort
cycling have been shown to increase mitochondrial enzyme
activity and protein content by 11–38% (9 –11, 21), which is
comparable with results from protocols consisting of 6 –10
sessions of 2 h duration at 60 –70% V̇O2 peak (13, 60). In
agreement with these studies suggesting increased capacity for
oxidative phosphorylation, we report a 14% increase in the rate
of PCr recovery in VL after 6 sessions of HIT (Fig. 4), which
suggests that the cumulative effects of repeated exercise sessions are required to promote an increased capacity for muscle
oxidative ATP production in vivo.
Several investigators have used the PCr recovery method to
examine the effect of endurance training on oxidative capacity
in vivo (19, 36, 47). Rates of PCr recovery have been shown to
increase by ⬃30% in response to training protocols involving
12–24 wk of aerobic exercise (36, 47). Our result is consistent
with a recent study reporting a similar increase in the rate of
PCr recovery (14%) in the quadriceps muscles of young adults
following the same 2-wk HIT protocol (19). A notable difference between our study and that of Forbes et al. (19) was the
experimental approach used to deplete intracellular PCr levels
for the oxidative capacity measure. We used a brief, maximal
contraction to recruit all muscle fibers, without inducing acidosis. Consequently, our results of enhanced PCr recovery
kinetics reflect global improvements in muscle oxidative capacity. In contrast, Forbes et al. (19) used moderate-intensity
contractions of the quadriceps muscles, which presumably
recruit a smaller portion of the muscle fibers (i.e., fibers
innervated by low-threshold motoneurons), and therefore, provides a measure of oxidative capacity that reflects a selective
population of muscle fibers. Despite the likely recruitment of
different proportions of muscle tissue, the increase in the rate
of PCr recovery following training was similar between studies, supporting the concept that HIT promotes mitochondrial
adaptations in all muscle fibers (22).
EFFECTS OF INTERVAL TRAINING ON MUSCLE ENERGETICS IN HUMANS
Effects of HIT on Vrest
As a surrogate for exercise training, chronic low-frequency
stimulation of isolated cat gracilis muscle has been shown to
increase resting and maximal oxygen consumption (33). Lowfrequency stimulation also increased mitochondrial density,
indicating that greater mitochondrial content may increase both
resting and maximal oxygen consumption in skeletal muscle.
Kacarowsky-Bielesz et al. (37) showed that three sessions of
30 min of moderate-intensity cycling (70% of V̇O2 max) resulted
in increased VPi¡ATP (⬃18%) of the soleus-gastrocnemius
complex of healthy men and women. Whereas this result
suggests that a relatively small amount of exercise training
elicits a higher rate of ATP synthesis in resting skeletal muscle,
the study did not reveal whether increased VPi¡ATP in resting
muscle was accompanied by greater mitochondrial content or
increased oxidative capacity. Our results suggest that lowvolume HIT does not elevate VPi¡ATP in resting VL muscle
(Fig. 3), which is consistent with the results from Richards et
al. (54) who reported unaltered whole body energy expenditure
after an identical training protocol. In agreement with these
results, Gibala and colleagues (23) provided evidence to suggest that myofibrillar protein synthesis was not stimulated after
HIT, which is consistent with our result of unchanged VL CSA
after completion of the 2-wk protocol. Despite an increase in
oxidative capacity after the sixth training session, k= and
VPi¡ATP remained unchanged. These results suggest that the
muscular adaptations that occur in response to short-term HIT
have differential effects on in vivo measures of resting and
maximal rates of oxidative ATP synthesis in human skeletal
muscle. Our results are informed by recent studies that have
examined the effects of pronounced mitochondrial modifications on VPi¡ATP in vivo. Increased expression of PGC-1␣ in
mouse muscle has been shown to elicit a 2.4-fold increase in
mitochondrial density but only a 50 – 60% increase in VPi¡ATP
(14). By inhibiting mitochondrial complex I in rat muscle, van
den Broek et al. (65) used a different approach to investigate
the link between mitochondrial function and muscle energetics
in vivo. While complex I inhibition reduced the rate of PCr
recovery by 46%, both k= and VPi¡ATP were unaffected by this
type of intervention. Collectively, these results suggest that
mitochondrial modifications, in the form of exercise training,
transgenic manipulations, or dysfunction do not affect k= and
only have a small, if any, influence on VPi¡ATP.
Effect of HIT on Muscle Energy Metabolites
Sustained or repeated bouts of maximal muscle activity
result in metabolic perturbations that may alter the intracellular
concentrations of energy metabolites in resting muscle (26, 29,
30, 61). Based on biopsy data, Gibala et al. (23) reported a
⬃16% reduction in muscle [ATP] 3 h after a single session of
HIT. Our study extends these findings by showing a similar
reduction (⬃21%) in intracellular [ATP] in vivo 15 h after the
first HIT session (Table 2). Consistent with results from a
biopsy study using an identical 2-wk HIT protocol (9), we
observed lower intracellular [ATP] in resting muscle after the
sixth training session (Table 2). Others have reported that
muscle [ATP] remains depressed even after 7– 8 wk of HIT
(10, 26, 30, 61). High rates of ATP turnover, as occurred
during each training session, result in elevated [AMP], increased flux through AMP deaminase, and thus accumulation
of inosine monophosphate (IMP) (29, 30). Subsequent breakdown of IMP to inosine and hypoxanthine can cause a loss of
adenine nucleotides from the muscle (29, 30). While restoration of purine nucleotide levels in the muscle is a slow process
and can last several days (10, 29), lower [ATP] in response to
HIT does not seem to be associated with muscle damage or
decreased exercise performance (30, 61). This interpretation is
supported by our observation of no change in muscle CSA in
response to training indicating no evidence of muscle swelling
in VL as would be expected in response to damage. Thus our
results are consistent with previous in vitro studies and provide, for the first time, evidence of a loss of cytosolic ATP in
vivo following a single and six sessions of HIT.
We calculated metabolite concentrations based on the assumption of stable concentration of total creatine (i.e., [PCr] ⫹
[Pi] ⫽ 42.5 mM). An alternate means of quantifying relative
metabolite concentrations based on the assumption that [ATP] ⫽ 8.2
mM was not used because similar training protocols have
resulted in a long-lasting decline in intracellular [ATP], with
no effects on concentrations of total creatine (30, 61). Furthermore, there is no evidence to suggest that HIT would affect the
stoichiometry between free creatine and inorganic phosphate.
Therefore, the assumption of stable [total creatine] appears to
be reasonable for quantification of metabolite levels in this type
of training study.
Control of oxidative phosphorylation in skeletal muscle,
particularly in vivo, appears to occur as a result of a combination of complex interactions (35, 58). It is generally understood that products of ATP hydrolysis (ADP, Pi) play important roles in regulating the rate of mitochondrial ATP synthesis. Consistent with the notion that [ADP] is a primary
regulator of oxidative phosphorylation in vivo (7, 12, 17), our
finding of a trend toward lower [ADP] in resting muscle
(calculated based on the creatine kinase equilibrium) suggests
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a primary regulator of oxidative phosphorylation, was unchanged after the first session. Notably, Gibala et al. (23)
showed that a single session of HIT was accompanied by
activation of AMP-activated protein kinase (AMPK), while
phosphorylation of protein kinase B/Akt tended to decrease in
response to exercise. These results led the authors to suggest
that HIT provides a concentrated stimulus for metabolic adaptations (i.e., mitochondrial protein synthesis) with no effect on
muscle growth (i.e., myofibrillar protein synthesis) (23). Considering that mitochondrial protein represents a small fraction
(⬃10%) of the total muscle protein pool (15) and the notion
that activation of AMPK conserves ATP by downregulating
anabolic pathways (25), it is possible that low-volume HIT
does not provide sufficient stimulus for increasing net muscle
protein turnover or other processes coupled to ATP hydrolysis.
To our knowledge, no studies have examined the effects of
low-volume HIT on rates of muscle protein synthesis and
breakdown. The 15-h time point was chosen to standardize
conditions (i.e., meals and a 10-h overnight fast) before muscle
metabolic testing and to minimize transitory effects (e.g., due
to elevated temperature and altered hormone levels) of the
preceding training session on muscle metabolism. Thus it is
possible that a shorter time delay between exercise and muscle
metabolic testing would have elicited a different response in
VPi¡ATP.
R339
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EFFECTS OF INTERVAL TRAINING ON MUSCLE ENERGETICS IN HUMANS
Saturation-Transfer Technique
Several studies have used the saturation transfer technique to
assess mitochondrial ATP production under various conditions, which has led to the concept of VPi¡ATP being an
important variable in the context of mitochondrial function and
health (37, 50, 51, 64). However, as recently reviewed by
several research groups (20, 38), there are some limitations
associated with using the saturation transfer technique to infer
information about the rate of oxidative ATP production in
resting skeletal muscle (38, 59, 65). First, the Pi¡ATP rate can
be influenced by ATP synthesis from nonoxidative sources.
Whereas net ATP production from glycolysis is negligible in
resting skeletal muscle, the glycolytic enzymes glyceraldehyde-3-phosphate dehydrogenase and phosphoglycerate kinase
catalyze a coupled, near-equilibrium reaction that may contribute to the Pi¡ATP exchange (8, 20, 59).
Second, particularly at low rates of respiration when ATP
synthase operates near equilibrium (i.e., low ADP/ATP), mitochondrial Pi↔ATP exchange may contribute to the Pi¡ATP
rate, such that the unidirectional rate of Pi¡ATP exchange
exceeds net ATP synthesis (20, 39, 59). These additional
sources of Pi¡ATP exchange likely contribute to the apparent
overestimation of oxidative ATP synthesis measured by the
saturation transfer technique in comparisons with other measures (e.g., oxygen consumption) of oxidative energy metabolism in resting skeletal muscle (20, 38).
Unfortunately, we cannot decipher the contribution from
glycolytic sources to our estimate of VPi¡ATP. However, because energy intake and macronutrient composition were
matched before each saturation transfer experiment, and the
participants were fasted for 10 h before these experiments, we
assume unaltered activation of glycolysis in resting muscle and
therefore do not expect that contributions from glycolytic
enzymes varied across time points. In contrast, we observed
increased ADP/ATP (Table 2), which moves the ATP synthase
reaction away from equilibrium, and presumably would result
in reduced mitochondrial Pi↔ATP exchange and a lower k=
after training (20, 38). Thus, in addition to the aforementioned
limitations of the saturation transfer experiment, it is possible
that exercise-induced alterations in the intracellular metabolic
environment constrain our ability to detect net changes in
mitochondrial ATP turnover using the saturation transfer tech-
nique. While we have demonstrated very good reproducibility
of the saturation transfer measure, additional studies are
needed to clarify the determinants of the Pi¡ATP rate and
examine the influence of altered intracellular metabolic state on
VPi¡ATP. In the meantime, our results show that the Pi¡ATP
rate, which is often used as a measure of in vivo mitochondrial
activity in resting skeletal muscle, is unchanged following HIT.
Perspectives and Significance
This study reveals that HIT promotes short-term (following
6 sessions) effects on the functional oxidative capacity of
muscle in vivo but causes no acute (single session) effect. At
the same time, the Pi¡ATP rate in resting muscle was unchanged after single or multiple training sessions. These novel
results indicate that repeated HIT sessions are required to
promote the relatively rapid adaptations that are responsible for
improved muscle oxidative capacity in vivo and suggest that
the mechanisms that regulate adaptations to maximal muscle
oxidative capacity are distinct from those regulating ATP
synthesis in resting tissue. This study expands our understanding of muscle remodeling after HIT by providing evidence of
the functional and temporal changes in human skeletal muscle
energetics in vivo, which have implications for developing
training interventions designed to improve skeletal muscle
function and health.
ACKNOWLEDGMENTS
The authors thank the participants for their enthusiasm and perseverance
throughout the study. We also thank Dr. John Buonaccorsi for statistical
advice, Logan Maynard for help with data collection, and all other members of
the Muscle Physiology Laboratory for help with various aspects of the project.
GRANTS
This work was supported by NIH/NIA K02 AG023582, the Keck Foundation, and a UMass Graduate School Fellowship.
DISCLOSURES
No conflicts of interest, financial or otherwise, are declared by the author(s).
AUTHOR CONTRIBUTIONS
Author contributions: R.G.L., D.E.B., and J.A.K.-B. conception and design
of research; R.G.L. performed experiments; R.G.L. analyzed data; R.G.L.,
D.E.B., and J.A.K.-B. interpreted results of experiments; R.G.L. prepared
figures; R.G.L. drafted manuscript; R.G.L., D.E.B., and J.A.K.-B. edited and
revised manuscript; R.G.L., D.E.B., and J.A.K.-B. approved final version of
manuscript.
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